AR-12 is an IND approved, COX-2 inhibitor derived drug that affects pathogen host cells by primarily upregulating autophagy. In vitro, AR-12 has shown broad spectrum efficacy on several bacterial strains including S. typhimurium, F. tularensis (Schu S4, LVS), and F. novicida. In vivo, AR-12 given i.v. reduced organ bacterial tenfold compared to untreated controls, but did not prevent host death due to typhoid fever. AR-12 concentrations were limited with in vivo application because the of the drug's hydrophobicity. To overcome solubility issues, we propose encapsulating AR-12 in acetalated dextran (Ac-DEX) particles that passively target the host cell. Ac-DEX is an acid sensitive polymer with tunable release kinetics that will release drug in the phagocyte's phagosome, due to the lower pH present. There are three aims for the R21 portion of this proposal. The first aim is to manufacture and characterize two types of Ac-DEX particles that encapsulate AR- 12: 1) a nanoparticle (NP) for i.v. or i.p. injection that is at the ideal size for passively targeting macrophages (500-1,000 nm); 2) a porous microparticle (PMP) that is ideal size (5-15 mm) for inhalation via the nose (i.n.) and penetration to the nasal associated lymphoid tissue (NALT). These particles will be manufactured, imaged through scanning electron microscopy, and characterized for drug loading through fluorescence spectroscopy. The second aim is to evaluate the Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) of unencapsulated AR-12 against drug susceptible and multidrug resistant M. tuberculosis (MDR TB) and encapsulated AR-12 against TB, F. tularensis (Schu S4), and S. typhimurium in human macrophages. Macrophages will be infected with one of three bacteria and treated with AR-12. Cell associated bacteria and CFUs will be evaluated to determine both the MIC and MBC. The MIC and MBC determined for F. tularensis will be used in Aim 3 for in vivo treatment with encapsulated AR-12 administered i.v. and i.p. with NPs, and i.n. with PMPs. A dosing study will be used to identify the best route and dosage (MIC, MBC, and 10 x MBC) by evaluating organ CFUs when administered at a comparable timing and frequency to traditional antibiotic regimens. The optimum route and dose will be used for an in-depth treatment evaluation of organ CFUs, histopathology, blood cytokine levels and survival. The milestones for progress to the R33 portion of the grant will be 1) encapsulation of AR-12 in Ac-DEX particles; 2) reduced macrophage associated M. tuberculosis with AR-12 treatment; 3) Increased survival and decreased organ bacterial load with AR-12 treatment of F. tularensis, in vivo. The R33 phase of this grant also has 3 specific aims. Aim 4 and 5 are to evaluate encapsulated AR-12 treatment in vivo against S. typhimurium, and TB, respectively in a manner similar to Aim 3. Aim 6 is to evaluate the immunotoxicity of encapsulated AR-12 and to progress encapsulated AR-12 towards IND approval. These studies will help to develop and characterize a new broad spectrum antibiotic and delivery platform that targets host cells.